ARTICLE https://doi.org/10.1038/s42003-019-0565-5 OPEN Regional heterogeneity impacts gene expression in the subarctic zooplankter Neocalanus flemingeri in the northern Gulf of Alaska Vittoria Roncalli 1,2, Matthew C. Cieslak1, Martina Germano1, Russell R. Hopcroft3 & Petra H. Lenz1 1234567890():,; Marine pelagic species are being increasingly challenged by environmental change. Their ability to persist will depend on their capacity for physiological acclimatization. Little is known about limits of physiological plasticity in key species at the base of the food web. Here we investigate the capacity for acclimatization in the copepod Neocalanus flemingeri, which inhabits the Gulf of Alaska, a heterogeneous and highly seasonal environment. RNA-Seq analysis of field-collected pre-adults identified large regional differences in expression of genes involved in metabolic and developmental processes and response to stressors. We found that lipid synthesis genes were up-regulated in individuals from Prince William Sound and down-regulated in the Gulf of Alaska. Up-regulation of lipid catabolic genes in offshore individuals suggests they are experiencing nutritional deficits. The expression differences demonstrate physiological plasticity in response to a steep gradient in food availability. Our transcriptional analysis reveals mechanisms of acclimatization that likely contribute to the observed resilience of this population. 1 Pacific Biosciences Research Center, University of Hawai’iatMānoa, 1993 East-West Rd., Honolulu, HI 96822, USA. 2 Department of Genetics, Microbiology and Statistics, Facultat de Biologia, IRBio, Universitat de Barcelona, Av. Diagonal 643, 08028 Barcelona, Spain. 3 Institute of Marine Science, University of Alaska, Fairbanks, 120 O’Neill, Fairbanks, AK 99775-7220, USA. Correspondence and requests for materials should be addressed to V.R. (email: [email protected]) COMMUNICATIONS BIOLOGY | (2019) 2:324 | https://doi.org/10.1038/s42003-019-0565-5 | www.nature.com/commsbio 1 ARTICLE COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-019-0565-5 ver the past 50 years, large-scale latitudinal shifts in throughout the region from Prince William Sound to the outer communities have been documented in both terrestrial shelf of the Gulf of Alaska25. Here, we applied RNA-Seq tech- O 1–4 and aquatic ecosystems . While community regime nology to obtain global gene expression profiles of pre- shifts are correlated with an overall increase in average tem- adult N. flemingeri collected across the shelf and in Prince Wil- peratures, the proximate causes for observed changes are more liam Sound. Large variation in transcriptional physiology was complex: climate forcing leads to cascading effects that alter many observed among pre-adult N. flemingeri collected across the shelf abiotic and biotic factors, which in turn affect individual fitness and in Prince William Sound. Regional differences in the and create new sets of winners and losers5,6. However, some expression of genes associated with metabolism, response to stress communities appear to be resilient to climate variability and and development were consistent with gradients in chlorophyll a, predicting how communities might respond to change is an active which is an indicator of food levels. While the results indicate a area of investigation1,7,8. large capacity for physiological acclimatization in N. flemingeri, One approach has been to investigate adaptive capacity of key they also suggest that offshore individuals in the high-nutrient species to stressors associated with global climate change, such as low-chlorophyll (HNLC) region of the Gulf of Alaska were increases in temperature and ocean acidification9,10. Species with experiencing nutritional stress, thus interfering with lipid accu- genetic divergence among populations have the potential for mulation required for successful preparation for diapause. rapid evolution and resilience to climate change through natural selection acting on existing genetic variation, and immigration of 11,12 Results resilient genotypes following local extinction events . How- fl ever, replacement with novel genotypes may be less available to Study overview. Neocalanus emingeri were collected in early planktonic species, which are not only widely distributed, but also May, when most individuals are in the pre-adult developmental stage (copepodid CV) and preparing for diapause, which requires in constant motion as they drift over large distances within 26 oceanic currents13,14. Oceanic mesozooplankton, like copepods the accumulation of storage lipids (Fig. 1a) . Collections show little genetic differentiation within oceanic provinces, and occurred during a 1-week oceanographic cruise (early May 2015) sometimes even across multiple oceans15–17. Thus, these organ- from six locations spanning the inner to outer shelf along the isms may depend on phenotypic plasticity to adapt to a hetero- Seward Line in the northern Gulf of Alaska and two stations geneous and changing environment. within Prince William Sound (Fig. 1b and Table 1). Gene expression profiles were obtained for individual pre-adults (CV) Planktontic organisms can experience nonoptimal conditions = through much of their life in oceanic habitats where they can be collected at each station (n 18) using RNA-Seq (Supplementary advected over large distances15,18,19. How do these organisms Table 1). Functional analysis of gene expression patterns was compensate for suboptimal conditions physiologically, while compared with genetic distance between individuals from dif- maintaining their fitness? High-throughput sequencing technol- ferent stations and with environmental gradients. ogies are providing new opportunities to investigate this question in zooplankton. Transcriptional differences among individuals Environmental gradients in the northern Gulf of Alaska. can be quantified using RNA-Seq. Environment-mediated shifts Concurrent environmental monitoring revealed differences in the in gene expression have been well documented both in the – vertical structure of temperature and salinity across stations laboratory and in the field20 22. Relative gene expression in consistent with previously described inshore–offshore gradients combination with a functional analysis of the regulated genes (Fig. 2a, b). All stations showed some stratification caused by can inform how an organism is responding to its ambient temperature and/or salinity gradients, with the most shallow and environment. Here, we used this approach to examine the pronounced gradients (~10 m) occurring at PWSA and PWS2. transcriptional physiology of a marine zooplankter, the Average temperatures between 1 and 50 m ranged between 6 and copepod Neocalanus flemingeri, a high-latitude species with a 7.3 °C (Fig. 3a). Temperatures at 100 m were similar across sta- complex life history adapted to optimize synchronization with tions (6.5 °C) with the exception of GAK1 (<6 °C). The low a seasonally changing environment that is also spatially temperature at GAK1 correlated with the lowest salinities mea- heterogeneous. sured at this depth (Fig. 2b). In general, surface salinities were The northern Gulf of Alaska is characterized by steep gradients lower at nearshore stations (PWS2, PWSA, and GAK1) as would in the physical and chemical environments driven by patterns in be expected from the greater influence of freshwater inputs at circulation, salinity, winds, and macro- and micro-nutrients23. these stations. Salinity increased with distance from shore as Differences in the physical and chemical environment lead to shown by the GAK4, GAK9, and GAK14 conductivity profiles resource gradients across the shelf, which impact the community and average salinities in the upper 50 m across the six stations composition and abundances of phytoplankton, micro- (Figs. 2b and Fig. 3a). zooplankton, and mesozooplankton18,19,24. In this highly variable Chlorophyll a levels varied across stations (Fig. 2c, d). Highest environment, N. flemingeri is a biomass dominant in April–May chlorophyll a concentrations were measured in the upper 30 m in Table 1 Summary of spring 2015 collections of Neocalanus flemingeri copepodid stage CV individuals analyzed for whole- organism patterns of gene expression using RNA-Seq PWS2 PWSA GAK1 GAK4 GAK9 GAK14 Date May 7, 2015 May 7, 2015 May 10, 2015 May 10, 2015 May 6, 2015 May 5, 2015 CTD cast# 22 24 34 40 10 7 Time (h) 15:10 19:30 07:45 14:15 08:30 18:00 Latitude N 60° 32.1′ 60° 49.3′ 59° 50.7′ 59° 24.5′ 58° 40.8′ 57° 56.6 Longitude W 147° 48.2′ 147° 24′ 149° 28′ 149° 2.9′ 148° 21′ 147° 39′ Depth (m) 738 476 264 199 276 2720 CTD (conductivity, temperature, and depth) cast#: date and time of CTD cast (Alaska daylight time), latitude and longitude, and total depth are listed for each station. Collection of zooplankton by vertical net tow from 100 to 0 m at all stations 2 COMMUNICATIONS BIOLOGY | (2019) 2:324 | https://doi.org/10.1038/s42003-019-0565-5 | www.nature.com/commsbio COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-019-0565-5 ARTICLE a Depth (m) 10 CI–IV CV 50 NIII–VI NIII NI–II Adults 300 400 Adult >500 females Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov b 62° 61° 60° 60° 59° 59° 58° 58° 57° 57° –144° –154° –152° –150° –148° –146° Fig. 1 Neocalanus flemingeri life cycle and map of study site. a Diagram of the life cycle of N. flemingeri from December to November. Feeding stages (nauplii [NIII-NVI] and copepodids [CI-CV]), are shown in black, nonfeeding stages (adult females and males [CVI] and early nauplii [NI and NII]) in light gray. Green oval indicates period of increased production starting in late March/early April and approximate timing of phytoplankton bloom (late April—early/ mid May). Depths in meters (not to scale) indicated on the right. Modified from Lenz and Roncalli26. b Map showing locations and names of stations in Prince William Sound (PWS) and Gulf of Alaska (GAK). Curved arrows: Prince William Sound (top); off shelf Gulf of Alaska (bottom; depth > 2000 m). Light gray lines indicate depth contours. Inset shows map of Alaska with the location of sampling area indicated by the black box Prince William Sound. Chlorophyll a levels declined with low at GAK4, GAK9, and GAK14 (Fig.